Orthotopic Mouse Model for Glioblastoma

All animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of the National Cancer Center, Republic of Korea. Every animal was randomized by body weight before conducting experiments. For the orthotopic mouse model^{29 (link),47 (link)}, cells were first resuspended in DMEM/F-12 supplemented with B27, EGF (10 ng/mL), and bFGF (5 ng/mL), and then transplanted into the left striatum of 5-week-old female BALB/c nude mice by stereotactic injection. The injection coordinates were 2.2 mm to the left of the midline and 0.2 mm posterior to the bregma at a depth of 3.5 mm. For syngeneic orthotopic model, GL261 cells were injected into 5-week-old female C57BL/6 mice. The tumors were extracted, pooled for each experimental group, and mechanically disaggregated using stainless steel operating scissors. The brain of each mouse was harvested and fixed in 4% PFA. JZL184 (30 mg/kg; Tocris Biosciences) was orally administered daily. PD-1 antibody (10 mg/kg; Bioxcell) was administered once a week for 9 weeks through an intraperitoneal injection. Survival was analyzed using GraphPad PRISM software (version 7; GraphPad PRISM, La Jolla, CA, USA).